Fluorescent probes for chromosomal painting

ABSTRACT

The present invention relates to fluorescent probes which can be used in multicolor fluorescence in situ hybridization, and mainly chromosome painting. The probes intended for labeling a chromosome are such that they are composed of a set of DNA segments which are more represented in certain chromosome bands and which are obtained by IRS-PCR amplification from said chromosomes using PCR primers specific for the repeated and dispersed Alu and LINE DNA sequences. 
     The invention comprises, in addition, methods of producing said probes, multicolor FISH methods which can use said probes as well as diagnostic kits comprising them. 
     Finally, the invention comprises combinations of fluorophores and optical filters.

The present invention relates to chromosome painting and moreparticularly the fluorescent probes which can be used in methods such asthe FISH (“Fluorescence In Situ Hybridization”) method. The inventionalso relates to combinations of fluorophores and of optical filters.

In situ hybridization is a technique which makes it possible to detect aDNA (or RNA) sequence by means of a probe having a specific sequencewhich is homologous to that studied. It is based on the complementarityof the nucleotides (A/T, A/U, G/C) and it can be carried out underprecise physicochemical conditions on chromosome or tissue preparations.The result of the in situ hybridization process is the formation of ahybrid between a probe and a target. In situ hybridization includes adenaturing step and also a step for detecting the hybrid or the probewhich is carried out after the in situ hybridization of the probe to thetarget. The sample may adhere in the form of a layer to the surface ofthe slide and this sample may, for example, comprise or containindividual chromosomes or chromosomal regions which have been treated inorder to maintain their morphologies under denaturing conditions. In thecontext of fluorescence in situ hybridization, the probes are labeledwith a fluorophore and the hybridization is revealed by fluorescentlabeling.

The recent development of this technique allows the simultaneousvisualization, on the same preparation, of several probes each revealedby a different fluorophore. This technique, called multicolor FISH ormulti-FISH, has been made possible by the combination of filtersspecific for the wavelengths of emission of the different fluorescentmolecules ensuring the labeling with the aid of a computer-aided imagingcarried out by means of infrared-sensitive high-resolution cold CCDcameras (Schröck et al., 1996; Speicher et al., 1996).

The use of probes having a specific sequence homologous to a precisechromosomal sequence or a whole chromosome coupled with the potentialfor a multicolor fluorescent labeling makes it possible to developso-called chromosome painting techniques, that is to say to obtainchromosomes of different colors and thus to obtain, if desired, amulticolor complete karyotype. Karyotype is understood to mean thecharacteristic arrangement of the chromosomes of a cell in themetaphase.

Within the general meaning of the term, “labeling” is understood to meanan entity such as a radioactive isotope or a nonisotopic entity such asenzymes, biotin, avidin, steptavidin, digoxygenin, luminescent agents,dyes, haptens and the like. The luminescent agents, depending on thesource of excitation energy, may be classified into radioluminescent,chemoluminescent, bioluminescent and photoluminescent (includingfluorescent and phosphorescent) agents. The term “fluorescent” refers ingeneral to the property of a substance (such as a fluorophore) toproduce light when it is excited by an energy source such as ultravioletlight for example.

“Chromosomal paint probe” is understood to mean a probe or a probecomposition such as the probe composition of this invention, which issuitable for hybridizing, under hybridization conditions, with a targetwhich comprises a predetermined chromosome of a multichromosomal genome.If only a fraction of such a chromosome is present in the sample beingsubjected to such a hybridization with such a probe composition, thenthis fraction hybridizes and is identified. In practice, a painted probeof this invention may be mixed with a second, a third, and the like, soas to allow the labeling and the simultaneous detection of the two,three, and the like, predetermined chromosomes.

The visualization of all the 24 human chromosomes has been made possibleby the use of a labeling with a combination of fluorochromes. Forexample, in the case of the use of 5 different fluorophores, 31combinations of fluorophores can be obtained. By using this labelingprinciple and 24 DNA probes specific for each of the human chromosomes,it has been possible to visualize each chromosome differentially. Theattribution, by computer processing, of artificial colors to each of thecombinations of fluorophores thus makes it possible to color the 24human chromosomes differently.

Rapidly, the strong potentials of such a multicolor labeling haveallowed the analysis of chromosomal aberrations which were difficult todetect up until now by conventional cytogenetic techniques for labelingchromosomes in bands (Summer et al., 1971; Dutrillaux and Lejeune, 1971)(Giema staining, labeling with BrdU, and the like). The principle oflabeling of chromosomes in bands is based on the differences in theaverage base pair composition (richness in GC) between the bands and onthe differences in the compaction of the chromatin between thechromosome bands. Chromosome painting has proved to be a very usefultool for detecting interchromosomal aberrations such as translocations,amplified DNA sequences such as the homogeneously stained regions calledHSR(HSR for Homogeneously Staining Regions) or the excesses ofchromosomal materials such as the marker chromosomes or double-minutechromosomes. Intrachromosomal aberrations such as deletions andduplications will only be detected as a function of the size of theaberrations, if the latter affect the length of the chromosomes, whereaschromosomal inversions will not at all be detectable by this method.

The limits of the use of the current spectral karyotyping as such aredue to the fact that it does not make it possible to detect the natureof the chromosome bands involved in an inter- or intrachromosomalrearrangement. To do this, it is essential to couple this technique tothe more conventional one of chromosome bands (R or G labeling) such asDAPI counterstaining, Giemsa or propidium iodide staining for example.

The requirement to combine different techniques of course constitutes ahandicap in the analysis of chromosomal aberrations and, moreover, theuse of the FISH or multi-FISH method which combines the high cost of theapparatus and the instrumentation necessary for the visualization ofchromosome painting with the high cost of the probes specific for thechromosomes restricts the possibilities of this technique spreading toresearch laboratories or to diagnostic laboratories.

Paint probes currently available on the market (GIBCO-BRL, Oncor,Boehringer Manheim and the like) are obtained by DOP-PCR amplificationusing degenerate PCR primers of chromosomes or for fragments ofchromosomes isolated by cumbersome techniques such as chromosome sortingby flow cytometry or microdissection of chromosomes. The hybridizationof probes obtained by DOP-PCR does not generate chromosome bands on thechromosomes. The generation of chromosome bands was sought through thecreation of artificial bands along chromosomes. This creation ofartificial bands requires the use of cumbersome and expensivetechniques. Furthermore, it results in bands which are not knownreference marks in the field of cytogenetics.

Some others have described the use of chromosome paint probes obtainedby amplification of chromosomes by IRS-PCR (Interspersed RepeatedSequences) using primers specific for DNA sequences which are repeatedand dispersed in the genome, such as the Alu and LINE sequences. Thecombined use of LINE and Alu PCR primers for the amplification of humanchromosomes by ISR-PCR was previously proposed by Lichter et al., 1990.However, the labeling in R bands which was obtained by the latter doesnot make it possible to ensure complete painting covering all theregions of the genome, in particular the telomeric regions and certain Gchromosome bands.

The object of the present invention is to provide chromosomal probeswhich can be obtained inexpensively and which, in addition, make itpossible to cause quality chromosome bands to appear directly onchromosomes painted in their entirety.

To do this, the present invention relates to probes intended for thelabeling of a chromosome, characterized in that they are composed of aset of DNA segments which are more represented in certain chromosomebands and which are obtained by IRS-PCR amplification from saidchromosomes with the aid of primers specific for the Alu and LINE DNAsequences.

The term “probe” refers to a polynucleotide or a mixture ofpolynucleotides such that DNA segments or DNA sequences are chemicallycombined with labeled individual entities. Each of the polynucleotidesconstituting a probe is characteristically in single-stranded form atthe time of hybridization to the target.

The term “DNA fragment”, “DNA segment” generally indicates only aportion of the polynucleotide or of a sequence present in a chromosomeor a chromosome portion. A polynucleotide for example may be cut orfragmented into a multitude of segments or of fragments. A chromosomecharacteristically contains regions which have DNA sequences containingrepeated DNA segments. The term “repeated” refers to the fact that aparticular DNA segment is present many times (at least twice), dispersedor otherwise in the genome. The so-called IRS-PCR method using primerswhich hybridize with the dispersed repeated sequences of the genome,such as, for example, the Alu or LINE sequences.

“Genome” designates the complete and unique copy of the geneticinstructions of an organism encoded by the DNA of this organism. In thepresent invention, the particular genome considered is multichromosomalsuch that the DNA is distributed in the cell between several individualchromosomes. The human genome is composed of 23 pairs of chromosomes, ofwhich an XX or XY pair determine the sex.

The term “chromosome” refers to the support for the genes carryingheredity in a living cell which is derived from chromatin and whichcomprises DNA and protein components (essentially histones). Theconventional international system for identifying and numbering thechromosomes of the human genome is used here. The size of an individualchromosome may vary within a multichromosomal genome and from one genometo another. In the present case of the human genome, the total length ofDNA of a given chromosome is generally greater than 50 million bp. Byway of comparison, the total length of the human genome is 3×10⁹ bp.

The genome of mammals contains repeated DNA sequences dispersed over thewhole genome. In humans, the majority of this type of sequences isrepresented by the different families of Alu sequences, which are about10⁶ in number and have in common a consensus sequence of 300 bp. Therepeated LINES (or L1) sequences are, like the Alu sequences, widelydistributed over the whole genome. They are nevertheless less numerous(about 10⁴). Their consensus sequence is about 6 kb. They are preferablysituated in the dark G bands (positive G or negative R), whereas the Alusequences are instead situated in the dark R bands (positive R)(Korenberg and Kirowski, 1988).

The DNA segments amplified by IRS-PCR according to the invention mayhave as source somatic hybrids, preferably rodent-human somatic hybrids,chromosomes or fragments of chromosome. The chromosomes or fragments ofchromosome may be obtained by chromosome sorting by flow cytometry or bychromosome microdissection. Within the framework of the preferredembodiment of the present invention, the DNA segments amplified byIRS-PCR have as source mono-chromosomal rodent-human somatic hybrids.

The DNA segments amplified by IRS-PCR according to the invention aremore represented in one type of cytogenetic band. The preferredcytogenetic bands are the G bands or the R bands. In the preferredembodiment of the present invention, the DNA segments amplified byIRS-PCR are more represented in R bands.

Repeated sequences, analogous to the human repeated sequences, are alsofound in the genome of rodents. However, the divergence of this type ofsequences between humans and rodents is sufficiently important for thereto be little homology between them. This divergence allows a selectiveamplification of DNA segments contained in the human chromosome whenhuman-rodent hybrids are used. Thus, starting with the DNA of ahuman-rodent somatic hybrid and using primers specific for the Aluand/or L1 consensus sequence, it is possible to selectively amplify byPCR the DNA sequences between two repeated sequences (“inverted”position) separated by a distance <5 kb. The product of amplificationthus obtained consists of a set of fragments (whose size variesapproximately from 100 bp to 5 kb) which is representative ofpractically the entire human chromosome contained in the DNA of thesomatic hybrid.

In general, the present invention is of course more particularlyintended for producing probes specific for human chromosomes, althoughit is possible to envisage chromosome paintings for other cell types(Sabile et al., 1997).

The probes of the present invention are characterized in that the probesare derived from a mixture of two IRS-PCR amplification products whichis composed of:

-   -   PCR amplification product obtained using the primer specific for        the Alu DNA sequences,    -   PCR amplification product obtained using the primer specific for        the Alu DNA sequences and the primer specific for the LINE DNA        sequences.

The present invention relates to a method of producing probes intendedfor labeling human chromosomes, characterized in that said methodcomprises the mixing of two amplification products obtained by twoIRS-PCR amplifications from said chromosomes using, on the one hand, PCRprimers specific for the Alu and LINE DNA sequences, and, on the otherhand, PCR primers specific for the Alu DNA sequences.

Any primer specific for the Alu or LINE sequences may be used in thepresent invention. Preferably, the primers specific for the Alu DNAsequences consist of the SR1 primer whose sequence is described in SEQID No 1 and the primer specific for the LINE DNA sequence is preferablythe L1H primer whose sequence is described in SEQ ID Nos 2 and 3.

Alternatively, probes according to the present invention may also bederived from a mixture of two IRS-PCR amplification products which iscomposed of:

-   -   PCR amplification product obtained using the primer specific for        the LINE DNA sequences,    -   PCR amplification product obtained using the primer specific for        the Alu DNA sequences and the primer specific for the LINE DNA        sequences.

The present invention also relates to a method of producing probesintended for labeling human chromosomes, characterized in that saidmethod comprises the mixing of two amplification products obtained bytwo IRS-PCR amplifications from said chromosomes using on the one handPCR primers specific for the Alu and LINE DNA sequences, and, on theother hand, PCR primers specific for the LINE DNA sequences.

The present invention also comprises the use of fluorophores and offilters whose combination makes it possible to ensure a chromosomepainting providing very readable karyotypes, that is to say to obtaincontrasted and well-defined chromosome paint colors.

Thus, the DNA probes described above are labeled directly or indirectlyby fluorescence techniques. Non-exhaustively, the fluorophores used forthe labeling may be chosen from markers of the cyanine, rhodamine,fluorescein, Bobipy, Texas Red, Oregon Green, Cascade Blue type. Inparticular, all the fluorophores cited in “Handbook of fluorescentprobes and research chemicals” (Richard P Haugland, 1996, MolecularProbes, MTZ Spence Ed., more particularly p 145–146, 153, 155–156,157–158, 161) can be used to label the probes of the present invention.

Preferably, the probes according to the invention are labeled with atleast 1, 2, 3, 4 or 5 fluorophores chosen from the following group:fluorescein isothiocyanate (FITC), Texas Red (TR for Texas Red), cyanine3 (Cy3), cyanine 5 (Cy5), cyanine 5.5 (Cy5.5), cyanine 7 (Cy7), Bodipy630/650.

The preferred method for labeling the DNA probes is “Nick translation”.However, the labeling may also be carried out by all the standardreactions for synthesis of DNA catalyzed by a polymerase and forlabeling oligonucleotides. For example, the labeling may be carried outby the techniques of random priming, amplification or primer extensionin situ.

The term “directly labeled probe” designates or describes a nucleic acidprobe whose labeling after the formation of hybrid with the target isdetectable without subsequent reagent treatment of the hybrid. Theprobes using the FITC, Texas Red, Cy3 and Cy5 fluorophores according tothe present invention are directly labeled.

The term “indirectly labeled probe” designates or describes a nucleicacid probe whose labeling after the formation of hybrid with the targetmust undergo an additional reagent treatment with one or more reagentsin order to combine therewith one or more entities from which adetectable compound finally result(s). For example, the probes may belabeled with DNP, digoxigenin or biotin and the revealing comprisesbringing the probe into contact with an anti-DNP or anti-digoxigeninantibody labeled with a fluorophore or with an avidin coupled to afluorophore. The probes using the fluorophores Cy7, Bodipy 630/650,Cy5.5. according to the present invention are indirectly labeled.

Preferably, the probe composition of the present invention comprises thelargest possible number of “directly labeled” probes. In addition to thefact that the directly labeled probes are easier to use, they allowbetter resolution. This good resolution is important for goodobservation of the chromosome bands. Preferably, the probe compositionof the present invention is of the “direct labeling” type for all thefluorophores. A preferred probe composition of the present invention isof the “direct labeling” type for 4 of the 5 fluorophores used. Inanother preferred composition, the directly labeled probes represent 3probes out of 5 or 6 fluorophores used.

The present invention also relates to a set of probes intended forlabeling human chromosomes, characterized in that it contains probesaccording to the present invention for each of the human chromosomes andfor a number of them. This set of probes will make it possible toanalyze in a single operation a complete karyotype so as to detecttherein and to identify therein possible chromosomal aberrations asdescribed above.

The present invention also relates to a multicolor FISH method intendedfor studying the karyotype, characterized in that the DNA probes arelabeled with fluorophores and in that each fluorophore having a specificabsorption and emission wavelength is combined with a pair of opticalfilteres, one for absorption and one for emission.

The fluorophores are chosen such that the overlapping of the absorptionand emission spectra between the different fluorophores is minimal. Moreparticularly, it is important that there is no overlapping between theabsorption and emission maxima of the different fluorophores.

Each fluorophore is used with a pair of optical filters; one forabsorption and one for emission. The filters make it possible to selectthe passbands such that the wavelengths corresponding to an overlap withanother fluorophore are eliminated. Accordingly, the filters used in thepresent invention are preferably of the narrow passband optical filtertype. The filters are preferably of a superior quality because it isimportant that the filter does not allow light outside the passband topass through.

Preferably, the present invention also relates to a multicolor FISHmethod intended in particular for studying the karyotype, characterizedin that the DNA probes according to the present invention are labeledwith fluorophores and in that each fluorophore having a specificabsorption and emission wavelength is combined with a pair of opticalfilters, one for absorption and the other for emission, said methodusing fluorophores and pairs of filters chosen from the following group:

-   -   a) the fluorophore FITC having a maximum absorption wavelength        of 494 nm and a maximum emission wavelength of 517 nm combined        with an excitation filter of the 490DF30 type (Omega Optical)        and with an emission filter of the 530DF30 type (Omega Optical),    -   b) the fluorophore Cy3 having a maximum absorption wavelength of        554 nm and a maximum emission wavelength of 568 nm combined with        an excitation filter of the 546DF10 type (Omega Optical) and        with an emission filter of the 570DF10 type (Omega Optical),    -   c) the fluorophore TR having a maximum absorption wavelength of        593 nm and a maximum emission wavelength of 613 nm combined with        an excitation filter of the 590DF10 type (Omega Optical) and        with an emission filter of the 615DF10 type (Omega Optical),    -   d) the fluorophore Cy5 having a maximum absorption wavelength of        652 nm and a maximum emission wavelength of 670 nm combined with        an excitation filter of the 650DF20 type (Omega Optical) and        with an emission filter of the 670DF10 type (Omega Optical),    -   e) the fluorophore Cy7 having a maximum absorption wavelength of        743 nm and a maximum emission wavelength of 767 nm combined with        an excitation filter of the 740DF25 type (Omega Optical) and        with an emission filter of the 780EFLP type (Omega Optical),    -   f) the fluorophore Cy5.5 having a maximum absorption wavelength        of 675 nm and a maximum emission wavelength of 694 nm combined        with an excitation filter of the 680DF20 type (Omega Optical)        and with an emission filter of the 700EFLP type (Omega Optical),    -   g) the fluorophore Bodipy 630/650 having a maximum absorption        wavelength of 632 nm and a maximum emission wavelength of 658 nm        combined with an excitation filter of the 630DF20 type (Omega        Optical) and with an emission filter of the 650DF10 type (Omega        Optical).

The invention also relates to a multicolor FISH diagnostic kitcharacterized in that it comprises DNA probes according to the presentinvention labeled with fluorophores and in that each fluorophore havinga specific absorption and emission wavelength is combined with a pair ofoptical filters, one for absorption and one for emission, said kit usingfluorophores and pairs of filters chosen from the following group:

-   -   a) the fluorophore FITC having a maximum absorption wavelength        of 494 nm and a maximum emission wavelength of 517 nm combined        with an excitation filter of the 490DF30 type (Omega Optical)        and with an emission filter of the 530DF30 type (Omega Optical),    -   b) the fluorophore Cy3 having a maximum absorption wavelength of        554 nm and a maximum emission wavelength of 568 nm combined with        an excitation filter of the 546DF10 type (Omega Optical) and        with an emission filter of the 570DF10 type (Omega Optical),    -   c) the fluorophore TR having a maximum absorption wavelength of        593 nm and a maximum emission wavelength of 613 nm combined with        an excitation filter of the 590DF10 type (Omega Optical) and        with an emission filter of the 615DF10 type (Omega Optical),    -   d) the fluorophore Cy5 having a maximum absorption wavelength of        652 nm and a maximum emission wavelength of 670 nm combined with        an excitation filter of the 650DF20 type (Omega Optical) and        with an emission filter of the 670DF10 type (Omega Optical),    -   e) the fluorophore Cy7 having a maximum absorption wavelength of        743 nm and a maximum emission wavelength of 767 nm combined with        an excitation filter of the 740DF25 type (Omega Optical) and        with an emission filter of the 780EFLP type (Omega Optical),    -   f) the fluorophore Cy5.5 having a maximum absorption wavelength        of 675 nm and a maximum emission wavelength of 694 nm combined        with an excitation filter of the 680DF20 type (Omega Optical)        and with an emission filter of the 700EFLP type (Omega Optical),    -   g) the fluorophore Bodipy 630/650 having a maximum absorption        wavelength of 632 nm and a maximum emission wavelength of 658 nm        combined with an excitation filter of the 630DF20 type (Omega        Optical) and with an emission filter of the 650DF10 type (Omega        Optical).

The filters according to the present invention are preferably such that:

-   -   they are of the 6-cavity type,    -   they have an ADI of 0°;    -   they have a tolerance λ₀±20% of FWHM,    -   they have a tolerance on FWHM of ±20% of FWHM,    -   they have an OD5 out-of-passband rejection for UV at 1200 nm    -   they have a transmission curve T≧50% at λ₀.

Preferably, the filters must also have a centered useful diametergreater than 21 mm, and a thickness ≦7 mm.

The fluorophores and the above filters may be used for the labeling ofthe probes according to the present invention or alternatively fordifferent probes used, for example, for chromosome painting or formulticolor FISH.

In the present invention, “filters” is understood to mean narrowpassband interference filters which transmit light within a given verynarrow spectral band centered around the reference wavelength λ₀. Theyare characterized by their transmission curve: T=f(λ). The width of theband is defined by the full width at half maximum transmission (FWHM for“Full Width at Half Maximum transmission”).

Outside the passband, the filter allows a residual signal which is asattenuated as possible to pass through.

The interference filters function on the principle of constructive anddestructive interference. The basic component of an interference filteris called cavity. It has two stacks of reflectors separated by a layerof a dielectric solid. The higher the number of cavities, the morerectangular the shape of the transmission curve (that is to say thegreater the slope of this curve). Moreover, the higher the number ofcavities, the better the coefficient of attenuation outside thepassband.

For the multifluorescence application, the excitation or emissionspectra of the fluorochromes used are very close to each other. It istherefore necessary to recover the minimum amount of signal possibleoutside the passband. Accordingly, 6-cavity filters which offer the bestcharacteristics at this level were chosen.

The filters used preferably have the following specifications:

-   -   they are designed to be used at normal light incidence,    -   the tolerance on the centre wavelength (λ₀) is ±20% of the        passband, for example, for a filter with a passband of 10 nm, λ₀        will be defined with a tolerance of ±2 nm,    -   the tolerance on the width of the passband is ±20%,    -   the coefficient of transmission T of these filters is greater        than 50%,    -   the out-of-passband rejection of these filters is 50D for        ultraviolet at 1200 nm; this means that outside the passband,        the coefficient of transmission is 10⁻⁵, that is to say 0.001%.        For standard filters, the out-of-passband rejection is ensured        for wavelengths ranging from 0.8 λ₀ to 1.2 λ₀; for example, for        a filter of λ₀=620 nm, the out-of-passband rejection occurs only        between 500 and 740 nm. However, for the multifluorescence        application, fluorochromes are observed whose spectra extend        from 350 to 800 nm. Accordingly, filters were used whose        out-of-passband rejection is ensured for ultraviolet at 1200 nm.

Finally, the present invention relates to a labeling kit characterizedin that it comprises at least DNA probes as described above or a set ofprobes as mentioned above.

The present invention relates to a multicolor FISH diagnostic kit,characterized in that it comprises the DNA probes as described above ora set of DNA probes as mentioned above and a combination of filters andof fluorophores as described above.

The FISH or multi-FISH technique to which reference is or will be madeseveral times in the present description is in particular described inSpeicher et al., 1996; Schröck et al., 1996.

Other characteristics and advantages of the present invention willemerge on reading the examples below.

The combinations of fluorophores and of optical filters described in theinvention may be used in multiple techniques involving fluorescencemicroscopy. Indeed, the fluorophores described in the present inventionmay be used to label many molecules or structures. Nonexhaustively, saidfluorophores may be used to label polypeptides, antibodies, nucleicacids, phospholipids, fatty acids, sterol derivatives, membranes,organelles and many other biological macromolecules. The organelles maybe mitochondria, endoplasmic reticulum, Golgi apparatus and lysosomes.

The combination of fluorophores and of optical filters according to theinvention may be used to carry out FISH. In particular, this may allowthe simultaneous use of several probes. This combination may be used tostudy multiple aspects such as cell morphology, the cytoskeleton, cellreceptors, ion channels, neurotransmitters, the circulation of fluids,membrane fluidity, cell viability and proliferation, apoptosis,pinocytosis, endocytosis and exocytosis, transduction, pH and ionconcentrations (for example calcium, potassium, magnesium and zincconcentrations) (Richard P Haugland, 1996, Molecular Probes, MTZ SpendEd.). It may allow the study of expression and translation.

The invention therefore also relates to a combination of fluorophoreschosen from: fluorescein isothiocyanate (FITC), Texas Red (TR for TexasRed), cyanine 3 (Cy3), cyanine 5 (Cy5), cyanine 5.5 (Cy5.5), cyanine 7(Cy7), Bodipy 630/650.

Preferably, the combination of fluorophores comprises at least 2, 3, 4,5, 6 or 7 fluorophores chosen from: fluorescein isothiocyanate (FITC),Texas Red (TR for Texas Red), cyanine 3 (Cy3), cyanine 5 (Cy5), cyanine5.5 (Cy5.5), cyanine 7 (Cy7), Bodipy 630/650.

A combination of preferred fluorophores of the present inventioncomprises the following 5 fluorophores: fluorescein isothiocyanate(FITC), Texas Red (TR), cyanine 3 (Cy3), cyanine 5 (Cy5) and cyanine 7(Cy7).

Another preferred combination of fluorophores of the present inventioncomprises the following 6 fluorophores: fluorescein isothiocyanate(FITC), Texas Red (TR), cyanine 3 (Cy3), Bodipy 630/650, cyanine 5 (Cy5)and cyanine 7 (Cy7).

Another preferred combination of fluorophores of the present inventioncomprises the following 6 fluorophores: fluorescein isothiocyanate(FITC), Texas Red (TR), cyanine 3 (Cy3), Bodipy 630/650 and cyanine 5(Cy5).

The combinations of fluorophores according to the invention may beincluded in a multicolor FISH diagnostic kit.

The combinations of fluorophores according to the invention may be usedto label an entity chosen from polypeptides, antibodies, nucleic acids,phospholipids, fatty acids, sterol derivatives, membranes, organellesand biological macromolecules.

In addition, the invention relates to a combination of fluorophorescombined with a pair of optical filters chosen from:

-   -   a. the fluorophore FITC having a maximum absorption wavelength        of 494 nm and a maximum emission wavelength of 517 nm combined        with an excitation filter of the 490DF30 type (Omega Optical)        and with an emission filter of the 530DF30 type (Omega Optical),    -   b. the fluorophore Cy3 having a maximum absorption wavelength of        554 nm and a maximum emission wavelength of 568 nm combined with        an excitation filter of the 546DF10 type (Omega Optical) and        with an emission filter of the 570DF10 type (Omega Optical),    -   c. the fluorophore TR having a maximum absorption wavelength of        593 nm and a maximum emission wavelength of 613 nm combined with        an excitation filter of the 590DF10 type (Omega Optical) and        with an emission filter of the 615DF10 type (Omega Optical),    -   d. the fluorophore Cy5 having a maximum absorption wavelength of        652 nm and a maximum emission wavelength of 670 nm combined with        an excitation filter of the 650DF20 type (Omega Optical) and        with an emission filter of the 670DF10 type (Omega Optical),    -   e. the fluorophore Cy7 having a maximum absorption wavelength of        743 nm and a maximum emission wavelength of 767 nm combined with        an excitation filter of the 740DF25 type (Omega Optical) and        with an emission filter of the 780EFLP type (Omega Optical),    -   f. the fluorophore Cy5.5 having a maximum absorption wavelength        of 675 nm and a maximum emission wavelength of 694 nm combined        with an excitation filter of the 680DF20 type (Omega Optical)        and with an emission filter of the 700EFLP type (Omega Optical),    -   g. the fluorophore Bodipy 630/650 having a maximum absorption        wavelength of 632 nm and a maximum emission wavelength of 658 nm        combined with an excitation filter of the 630DF20 type (Omega        Optical) and with an emission filter of the 650DF10 type (Omega        Optical).

Preferably, the combination of fluorophores combined with a pair ofoptical filters comprises at least 1, 2, 3, 4, 5, 6 or 7 fluorophoresand filters chosen from:

-   -   a) the fluorophore FITC having a maximum absorption wavelength        of 494 nm and a maximum emission wavelength of 517 nm combined        with an excitation filter of the 490DF30 type (Omega Optical)        and with an emission filter of the 530DF30 type (Omega Optical),    -   b) the fluorophore Cy3 having a maximum absorption wavelength of        554 nm and a maximum emission wavelength of 568 nm combined with        an excitation filter of the 546DF10 type (Omega Optical) and        with an emission filter of the 570DF10 type (Omega Optical),    -   c) the fluorophore TR having a maximum absorption wavelength of        593 nm and a maximum emission wavelength of 613 nm combined with        an excitation filter of the 590DF10 type (Omega Optical) and        with an emission filter of the 615DF10 type (Omega Optical),    -   d) the fluorophore Cy5 having a maximum absorption wavelength of        652 nm and a maximum emission wavelength of 670 nm combined with        an excitation filter of the 650DF20 type (Omega Optical) and        with an emission filter of the 670DF10 type (Omega Optical),    -   e) the fluorophore Cy7 having a maximum absorption wavelength of        743 nm and a maximum emission wavelength of 767 nm combined with        an excitation filter of the 740DF25 type (Omega Optical) and        with an emission filter of the 780EFLP type (Omega Optical),    -   f) the fluorophore Cy5.5 having a maximum absorption wavelength        of 675 nm and a maximum emission wavelength of 694 nm combined        with an excitation filter of the 680DF20 type (Omega Optical)        and with an emission filter of the 700EFLP type (Omega Optical),    -   g) the fluorophore Bodipy 630/650 having a maximum absorption        wavelength of 632 nm and a maximum emission wavelength of 658 nm        combined with an excitation filter of the 630DF20 type (Omega        Optical) and with an emission filter of the 650DF10 type (Omega        Optical).

The combinations of fluorophores combined with the pair of opticalfilters according to the invention may be included in a multicolor FISHdiagnostic kit.

The combinations of fluorophores combined with a pair of optical filtersaccording to the invention may be used to label an entity chosen frompolypeptides, antibodies, nucleic acids, phospholipids, fatty acids,sterol derivatives, membranes, organelles and biological macromolecules.

The probes and combinations of fluorophores according to the inventionmay be used with any type of microscope (monochromator, laser,fluorescence microscope). Preferably, the invention uses a fluorescencemicroscope.

Various publications and patents are cited in the description. Thedisclosures contained in the publications and patents identified byreferences in this application are incorporated by way of reference intothe present application for a more detailed description of the contentof the present invention.

EXAMPLES

1. Preparation of the Probes

The genomic DNA extracted from various human-rodent somatic hybrid lines(NIGMS Human genetic Mutant Cell Repository, Coriell Institute forMedical Research, Camdem) (Table 1) served as template for the PCR.

TABLE 1 Chromosome Line reference  1 GM 13139  2 GM 10826B  3 GM 10253 4 GM 10115  5 GM 10114  6 GM 11580  7 GM 10791  8 GM 10156C  9 GM 1061110 GM 10926B 11 GM 10927A 12 GM 10868 13 GM 10898 14 GM 11535 15 GM11715 16 GM 10567 17 GM 10498 18 GM 11010 19 GM 10449 20 GM 13260 21 GM10323 22 GM 10888 X GM 6318B

The PCR was carried out either in the presence solely of the primer SR1(situated at the 3′ end of the Alu consensus sequence, position 241 to261: 5′ CCACTGCACTCCAGCCTGGG 3′ (SEQ ID No. 1) (Romana et al., 1993), orin the presence of the primer SR1 and of the primer L1H:

5′CATGGCACATGTATACATATGTAAC(A/T)AACC 3′ (SEQ ID No. 2 and No. 3)(Ledbetter et al., 1990). When only the primer SR1 was used in the PCR,the product of amplification labeled and used as probe on metaphasechromosomes was stained almost completely the corresponding chromosome[sic] (with the exception of the centromeric regions) with an R-typeband profile (as has been described by Lichter et al., 1990 with othertypes of Alu primers). However, in order to have a representation of thenegative R bands, a PCR was also carried out by incorporating the 2primers: SR1 and L1. Thus, when the 2 products of amplification (SR1 andSR1/L1) were mixed and used as probe, the negative R bands were indeedstained and the telomeric regions were perfectly delimited.

PCR Conditions

The PCR reaction took place in a final volume of 50 μl containing 500 ngof genomic DNA (somatic hybrid), 1 μM of each oligonucleotide (either 1μM SR1, or 1 μM SR1 and 1 μM L1H), 10 mM Tris-HCl, 50 mM KCl, 2.5 mMMgCl₂, 0.01% gelatin, 250 μM of each deoxynucleotide triphosphate (dATP,dGTP, dCTP, dTTP) and 2.5 U of Thermophilus aquaticus DNA polymerase(Perkin-Elmer-Cetus). The initial denaturation was carried out at 96° C.for 6 min, followed by 30 cycles: denaturation at 94° C. for 1 min,annealing at 63° C. for 1 min, extension at 72° C. for 10 min At the endof the cycles, a final extension at 72° C. for 10 min was carried out.

The 2 products of amplification (SR1 and SR1/L1H) were mixed and thenprecipitated with ethanol. The DNA pellet was taken up in 20 μl of waterand the DNA concentration was estimated on a 1.3% agarose gel.

Labeling of the Probes by “Nick Translation”

15 μg of the mixture of PCR products were able to be labeled during asingle Nick translation reaction. The reaction took place in a finalvolume of 500 μl, containing 20 μM of each of the deoxynucleotidetriphosphates (dATP, dGTP, dCTP), 10 μM of dTTP and 10 μM of modifieddUTP, 50 μM Tris-HCl, 5 μM MgCl₂, 1 μM β-mercaptoethanol and 20 U of amixture of enzymes (Dnase/Polymerase: Boehringer-Mannheim). To directlylabel the probe in fluorescence, the modified nucleotide was eitherdUTP-12-FITC (Boehringer-Mannheim) or dUTP-12-Texas Red (MolecularProbes) or dUTP-Cy3 (Amersham) or dUTP-Cy5 (Amersham). On the otherhand, for an indirect labeling, dUTP-16-biotinylated(Boehringer-Mannheim) was used. The labeling was carried out in anEppendorf tube (2 ml) overnight at 15–16° C. The free nucleotides werethen removed by precipitation of the probe with ethanol. The DNA pelletwas taken up in 500 μl of TE 10:1 (10 mM Tris-HCl, 1 mM EDTA, pH8) sothat the probe is at a concentration of about 30 ng/μl.

Composition of the Mixture of the 23 Chromosome Paint Probes

Each probe specific for a chromosome was labeled individually with thevarious modified dUTPs (fluorescent or otherwise). Depending on therichness in R and G bands of each of the chromosomes and depending ontheir size, those which had or otherwise to be composed of differentfluorochromes were established a priori. For example, combinations of 3or 4 fluorochromes were preferably used for chromosomes rich in R bands(e.g.: chromosome 19); on the other hand, for chromosomes low in Rbands, only 1 to 2 fluorochromes were used (e.g.: chromosome 8). Thischoice also depended, for each fluorochrome, on the combination ofexcitation and emission filters. Indeed, when a probe was labeled inequivalent proportion with different fluorochromes, the signalintensities for the different fluorochromes are not necessarilycomparable. That depends indeed on the quality of the excitation andemission filters for each fluorochrome, but also on the quantity offluorescence emitted by the fluorochrome. The latter itself depends onthe intensity of the excitation luminous flux and therefore on thespectral power of the light source (Mercury Lamp HBO 100 W−OSRAM). Amongall these parameters, it was preferably chosen to optimize the resolvingpower of the combinations of filters, in order to obtain on emission thebest signal/background noise ratio for each fluorochrome. Indeed, thedifferences in intensity of fluorescence may be compensated for, on theone hand, by increasing or decreasing the exposure time during theacquisition of the image by the camera (Hamamatsu C4880), but also byvarying the concentrations of probes depending on the fluorescent markerused. Finally, the concentrations of probes were adjusted such that fora given fluorochrome (and therefore for a given filter combination), allthe labeled chromosomes emit a fluorescence of equivalent intensity.

The composition of the 23 chromosome paint probes was therefore definedexperimentally and precisely after many control experiments (Table 2).

400 μg (about 27-fold) of Cot1 DNA (human competitor DNA) were added tothese 1470 ng of mixture of chromosomal probes (50 different probes).The DNA mixture was then precipitated with ethanol. The pellet was takenup in 10 μl of hybridization mixture (50% formamide, 10% dextransulfate, 2×SSC (pH 7), 1 μg/μl of sonicated herring sperm DNA).

TABLE 2 Fluor Mixture Chrom. FITC Cy3 TR Cy5 Bio/Cy7 1470 ng =  1 35 35 2 40 40  3 30 25 55  4 50 50  5 50 50  6 40 30 70  7 30 35 65  8 60 3090  9 30 20 50 10 30 25 20 75 11 30 40 70 12 35 50 85 13 60 45 105  1425 20 25 70 15 50 15 15 80 16 30 20 20 70 17 15 25 20 60 18 40 40 19 2510 20 20 75 20 20 20 30 70 21 30 30 60 22 10 15 20 45 X 30 30 60 Fullname of the fluorochromes: FITC: Fluorescein isothiocyanate; TR: TexasRed; Cy3: Cyanine 3; Cy5: Cyanine 5; Bio: Biotin; Cy7: Cyanine 7

An Alternative Preparation of the Probes

Alternatively, the preparation of the mixture of probes was carried outbefore their labeling by Nick translation for each fluorochrome (Table3). 5 mixtures of probes were thus obtained which were labeled by Nicktranslation with the aid of modified nucleotides according to thefollowing protocol.

1 to 2 μg of the mixture of probes were labeled by Nick translation in avolume of 50 μl containing: 20 μM of each of the deoxynucleotidetriphosphates (dATP, dGTP, dCTP), 10 μM of dTTP and 10 μM of modifieddUTP, 50 μM Tris-HCl, 5 μM MgCl₂ , 1 μM β-mercaptoethanol and 20 U of amixture of enzymes (Dnase/Polymerase: Boehringer-Mannheim). To directlylabel the probe in fluorescence, the modified nucleotide wasdUTP-12-FITC (Boehringer-Mannheim) or dUTP-12-Texas Red (MolecularProbes) or dUTP-Cy3 (Amersham) or dUTP-Cy5 (Amersham). On the otherhand, for an indirect labeling, dUTP-16-biotinylated(Boehringer-Mannheim) was used. The labeling was carried out in anEppendorf tube (2 ml) overnight at 15–16° C.

After labeling, the 5 mixtures of chromosomal probes (14.75 μg) wereprecipitated together (with ethanol) in the presence of 400 μg (about27-fold) of Cot1 DNA (human competitor DNA). The pellet was taken up in10 μl of hybridization mixture (50% formamide, 10% dextran sulfate,2×SSC (pH 7), 1 μg/μl of sonicated herring sperm DNA).

This alternative method of labeling has the advantage of making theprotocol for labeling the probes less cumbersome and simpler by reducingthe number of labelings by Nick translation to five instead of fifty andby preparing a single mixture of probes instead of two.

2. Fluorescence In Situ Hybridization

The procedure was that described by Cherif et al., 1990, with a fewmodifications.

Preparation of the Chromosomes in Metaphase

The preparation of the metaphase chromosomes was carried out startingwith a culture of circulating lymphocytes obtained by venous puncture ofa normal subject. The lymphocytes stimulated by phytohemagglutinin (PHA)(100 μl per 0.8 ml of culture) were cultured for 72 hours at 37° C. inRPMI-1640 medium. The cells were then synchronized by addingmethotrexate (10 μM) for 17 hours and then rinsed and recultured in thepresence of 5-bromodeoxyunridine (BrdU) (0.1 mM) for 6 hours. After theaction of colchicine (1 mg/ml) for 15 min, the disruption of the cellswas obtained, by resuspension in a hypotonic KCl solution (75 mM). Thechromosomes were fixed in a methanol/acetic acid mixture (3 vol/1 vol)and one to two drops of cellular suspension were spread on each slide.The slides, dried at room temperature for 2 to 3 days, were then storedat −20° C. for several months.

TABLE 3 Fluor. Chrom. FITC Cy3 TR Cy5 Bio/Cy7 Mixture  1 0.3 0.3  2 0.40.4  3 0.3 0.25 0.55  4 0.5 0.5  5 0.5 0.5  6 0.4 0.3 0.7  7 0.3 0.350.65  8 0.6 0.3 0.9  9 0.3 0.2 0.5 10 0.3 0.25 0.2 0.75 11 0.3 0.4 0.712 0.35 0.5 0.85 13 0.6 0.45 0.105 14 0.25 0.2 0.25 0.7 15 0.5 0.15 0.150.8 16 0.3 0.2 0.2 0.7 17 0.15 0.25 0.2 0.6 18 0.4 0.4 19 0.25 0.1 0.20.2 0.75 20 0.2 0.2 0.3 0.7 21 0.3 0.3 0.6 22 0.1 0.15 0.2 0.45 X 0.30.3 0.6 Total (μg) 3.7 2.7 2.75 3.15 2.45 14.75 Full name of thefluorochromes: FITC: Fluorescein isothiocyanate; TR: Texas Red; Cy3:Cyanine 3; Cy5: Cyanine 5; Bio: Biotin; Cy7: Cyanine 7

Preparation of the Slides

The slides were treated with RNase (100 μg/ml) for 1 hour at 37° C. andthen rinsed 3 times (5 min each) in a 2× SSC solution, pH 7. The slideswere then dehydrated by successive passes in a series of ethanol bathsat increasing concentration (70%, 80%, 90% and 100% and 2 min per bath),and dried. The chromosomal DNA was denatured by immersing the slides ina 70% formamide/2×SSC bath (pH 7) at 70° C. for 2 minutes. Thedenaturation was stopped by immersing the slides (2 minutes) in 70%ethanol cooled to −20° C. and kept in an ice bath. The slides were thendehydrated by successive passes in a series of ethanol baths atincreasing concentration and dried. The slides were then incubated for 8to 10 min at 37° C. in a solution (20 mM Tris-HCl, pH 7.4, 2 mM CaCl₂)containing proteinase K (100 ng/ml) and then dehydrated in a series ofethanol baths and dried.

Hybridization and Detection of the Signal

The mixture of probes and of competitor DNA (10 μl) was denatured for 10min at 70° C. and then immersed in ice and prehybridized for at least 3hours at 37° C. This mixture was then deposited on the slide and coveredwith a glass coverslip (18×22 mm). The hybridization took place over 2–3days at 37° C. in a humid chamber.

The slides were then washed in a series of 3 baths of 50% formamide, 2×SSC, pH 7 (3 min each) at a temperature of 42–45° C., followed by 5washes in 2× SSC, pH 7 (2 minutes each) and by a one minute wash in a 1×BN solution (0.1 M sodium bicarbonate, 0.05% nonidet P-40). Thebiotinylated probes were detected by adding avidin (Vector LaboratoriesBiosys) coupled to cyanine 7 (Amersham) (Avidin-Cy7) (5 μg/ml). Thecoupling of the avidin to cyanine 7 was carried out with a coupling kit(Amersham). To reduce the problems of background noise linked tononspecific bindings of avidin, the slides were previously incubated for10 minutes in a 1× BN solution containing 5% skimmed milk powder. Theslides were then incubated for ½ an hour at 37° C. in a solutioncontaining avidin-Cy7 (5 μg/ml in 1× BN+5% milk powder) and thensuccessively washed 3 times (2 minutes) in a 1× BN solution at 45° C.The fluorescent signal was amplified by adding a layer of biotinylatedanti-avidin antibodies (5 μg/ml) (Vector Laboratories, Biosys France),followed by a layer of avidin-Cy7 (5 μg/ml) according to the protocoldescribed by Pinkel et al., 1986. For each layer, the slides wereincubated for 30 minutes at 37° C. and then washed 3 times in a 1× BNsolution. The probes labeled with dUTP-FITC, dUTP-TR, dUTP-Cy3 anddUTP-Cy5 required no additional revealing step.

The slides were examined with the aid of an epifluorescencephotomicroscope (DMRX B, LEICA) equipped with the combination of filterswhich is described above. Two independent wheels were used to carry 8filters each. The wheel carrying the excitation filters was insertedimmediately after the Hg lamp and that carrying the emission filters wasplaced above the objectives and the beam separating filter. Before theacquisition of the images, 20 μl of an anti-fade solution (Johnson etal., 1981) were deposited on each slide and covered with a coverslip(anti-fade solution: 100 mg of PPD (p-phenylenediamine, Sigma) in asolution composed of 10 ml of PBS and 90 ml of glycerol; the pH of thesolution was adjusted to 8.0 with 0.1 M NaOH). The anti-fade solutionmakes it possible to avoid the rapid extinction of the fluorescenceemitted by the different fluorochromes when they are subjected to strongirradiation.

3. Example of Use of a Combination of 6 Fluorophores

Since cyanine 7 is a relatively unstable fluorochrome, efforts were madeto replace it with another fluorochrome, for example Bodipy 630/650(Molecular Probes). Coupled to an antibody or a molecule of avidin,Bodipy makes it possible to indirectly reveal a probe labeled withbiotin, with digoxigenin or with dinitrophenol (DNP). In this case, thechoice of the 5 fluorochromes for the multifluorescence can no longer bethe same because the absorption and emission spectra of cyanine 5 and ofBodipy 630/650 are too close for there to be good discrimination betweenthese two fluorochromes. The choice will be the following:

-   -   fluorescein isothiocyanate (FITC)    -   Texas Red (TR)    -   Cyanine 3 (Cy3)    -   Bodipy 630/650    -   Cyanine 5.5 (Cy5.5)

This choice also has the advantage of allowing the use of cyanine 7 as6th fluorochrome if need be (for example for applications where it isnot possible to carry out combinatory probe labeling, and where it wouldbe advantageous to have different probes which can be discriminatedbetween with a maximum of different fluorochromes).

In this case, the Cy5.5 should also be coupled to an antibody or amolecule of avidin in order to be able to reveal a probe labeled withbiotin, digoxigenin or DNP.

For example, for multifluorescence karyotyping, the different systemsfor labeling and revealing the probes will be:

Labeling Type of labeling Revealing fluorescein isothiocyanate (FITC)direct no Texas Red (TR) direct no Cyanine 3 (Cy3) direct no digoxigenin(Dig) indirect anti-dig-Bodipy 630/650 biotin (Bio) indirectavidin-Cy5.5

Should it be desirable to use 6 fluorochromes at the same time, thechoice would be:

Labeling Type of labeling Revealing fluorescein isothiocyanate (FITC)direct no Texas Red (TR) direct no Cyanine 3 (Cy3) direct nodinitrophenol (DNP) indirect anti-DNP Bodipy 630/650 digoxigenin (Dig)indirect anti-dig-Cy5.5 biotin (Bio) indirect avidin-Cy7

Although the preferred embodiments of the invention have beenillustrated and described, it should be considered that many changes maybe made by persons skilled in the art to depart [sic] from the spiritand scope of the present invention.

REFERENCES

-   Cherif D., Julier C., Delattre O., Derre J., Lathrop G M., Berger    R., (1990). Simultaneous localization of cosmids and chromosome    R-banding by fluorescence microscopy: Application to regional    mapping of chromosome 11. Proc. Natl. Acad. Sci. USA 87, 6639.-   Dutrillaux B., Lejeune J. (1971). Sur une nouvelle technique    d'analyse du caryotype humain (On a new technique for analyzing    human karyotype). C.R. Acad. Sci. 272, 2638.-   Johnson G. D., De Nogueira C., Aranjo J. G. M. (1981). A simple    method for reducing the fading of immunofluorescence during    microscopy. J. Immunol. Methods 43, 349.

Korenberg J. R., Rikowski M. C. (1988). Human genome organization: Alu,Lines and the molecular structure of metaphase chromosome bands. Cell53, 391.

-   Ledbetter S. A., Garcia-Heras J., Ledbetter D. H. (1990).    “PCR-karyotype” of human chromosomes in somatic cell hybrids.    Genomics 8, 614.-   Lichter P., Ledbetter S. A., Ledbetter D. H., Ward D. C. (1990).    Fluorescence in situ hybridization with Alu and L1 polymerase chain    reaction probes for rapid characterization of human chromosomes in    hybrid cell lines. Proc. Natl. Acad. Sci. USA 87, 6634.-   Pinkel D., Straume T., Gray J. W. (1996). Cytogenetic analysis using    quantitative, high sensitivity fluorescence hybridization. Proc.    Natl. Acad. Sci. USA 83, 2934.-   Richard P Haugland, 1996, Molecular Probes, MTZ Spence Ed.    <<Handbook of fluorescent probes and research chemicals >>-   Romana S. P., Tachdjian G., Druart L., Cohen D., Berger R.,    Cherif D. (1993). A simple method for prenatal diagnosis of trisomy    21 on uncultured amniocytes. Eur. J. Hum. Genet. 1, 245.-   SabileA., Poras I., Cherif D., Goodfellow P., Avner P. (1997).    Isolation of monochromosomal hybrid for mouse chromosomes 3, 6, 10,    12, 14 and 18. Mammalian Genome 8, 81.-   Schröck E., du Manoir S., Veldman T., Schoell B., Wienberg J.,    Ferguson-Smith M. A., Ning Y., Ledbetter D. H., Bar-Am I., Soenksen    D., Garini Y., Ried T. (1996). Multicolor spectral karyotyping of    human chromosomes. Science 273, 494–497.-   Speicher M. R., Ballard S. G., Ward D C. (1996). Karyotyping human    chromosomes by combinatorial multi-fluor FISH. Nature Genetics 12,    368–375.

Summer A. T., Evans H. J., Buckland R. A. (1971). A new technique fordistinguishing between human chromosomes. Nature (New Biol) 232, 31.

1. A method of producing probes intended for labeling human chromosomescomprising mixing first amplification products and second amplificationproducts obtained by two IRS-PCR amplifications from said chromosomes,labeling said amplification products with one or more fluorophore, anddetecting said labeled amplification products, wherein said firstamplification products are obtained using PCR primers specific for Aluand LINE DNA sequences and said second amplification products areobtained using PCR primers specific for Alu DNA sequences and saidamplification products are labeled and detected with: (a) thefluorophore Cy3 having a maximum absorption wavelength of 554 nm and amaximum emission wavelength of 568 nm combined with an excitation filterof the 546DF10 type (Omega Optical) and with an emission filter of the570DF10 type (Omega Optical); (b) the fluorophore TR having a maximumabsorption wavelength of 593 nm and a maximum emission wavelength of 613μm combined with an excitation filter of the 590DF10 type (OmegaOptical) and with an emission filter of the 615DF10 type (OmegaOptical); and (c) at least one fluorophore, absorption filter, andemission filter are selected from the group consisting of: (i) thefluorophore FITC having a maximum absorption wavelength of 494 nm and amaximum emission wavelength of 517 nm is coupled with the excitationfilter of the 490DF30 type (Omega Optical) and with an emission filterof the 530DF30 type (Omega Optical); (ii) the fluorophore Cy5 having amaximum absorption wavelength of 652 nm and a maximum emissionswavelength of 670 nm combined with an excitation filter of the 650DF20type (Omega Optical) and with an emission filter of the 670DF10 type(Omega Optical); (iii) the fluorophore Cy7 having a maximum absorptionwavelength of 743 nm and a maximum emissions wavelength of 767 nmcombined with an excitation filter of the 740DF25 type (Omega Optical)and with an emission filter of the 780EFLP type (Omega Optical); (iv)the fluorophore Cy5.5 having a maximum absorption wavelength of 675 nmand a maximum emission wavelength of 694 nm combined with an excitationfilter of the 680DF20 type (Omega Optical) and with an emission filterof the 700EFLP type (Omega Optical); and (v) the fluorophore Bodipy630/650 having a maximum absorption wavelength of 632 nm and a maximumemission wavelength of 658 nm used with an excitation filter of the630DF20 type (Omega Optical) and with an emission filter of the 650EFLPtype (Omega Optical).
 2. A method of identifying human chromosomescomprising performing a multicolor FISH analysis using a plurality ofprobes and hybridizing one or more human chromosomes with said pluralityof probes, said plurality of probes comprising a set of DNA segmentswhich are more represented in certain chromosome bands and which areobtained by IRS-PCR amplification from said chromosomes using primersspecific for Alu and LINE DNA sequences and said probes are labeled withone or more fluorophore, each of said one or more fluorophore having aspecific absorption and emission wavelength, wherein each of said one ormore fluorophore is used with a pair of optical filters one forabsorption and one for emission and wherein said fluorophore and pairsof filters are selected from the group consisting of: (a) thefluorophore FITC having a maximum absorption wavelength of 494 nm and amaximum emission wavelength of 517 nm used with the excitation filter ofthe 490DF30 type (Omega Optical) and with an emission filter of the530DF30 type (Omega Optical); (b) the fluorophore Cy3 having maximumabsorption wavelength of 554 nm and a maximum emission wavelength of 568nm used with an excitation filter of the 546DF10 type (Omega Optical)and with an emission filter of the 570DF10 type (Omega Optical); (c) thefluorophore TR having a maximum absorption wavelength of 593 nm and amaximum emission wavelength of 613 nm used with a excitation filter ofthe 590DF10 type (Omega Optical) and with an emission filter of the615DF10 type (Omega Optical); (d) the fluorophore Cy5 having a maximumabsorption wavelength of 652 nm and a maximum emissions wavelength of670 μm used with an excitation filter of the 650DF20 type (OmegaOptical) and with an emission filter of the 670DF10 type (OmegaOptical); (e) the fluorophore Cy5.5 having a maximum absorptionwavelength of 675 nm and a maximum emission wavelength of 694 nm usedwith an excitation filter of the 680DF20 type (Omega Optical) and withan emission filter of the 700EFLP type (Omega Optical); and (f) thefluorophore Bodipy 630/650 having a maximum absorption wavelength of 632nm and a maximum emission wavelength of 658 nm used with an excitationfilter of the 630DF20 type (Omega Optical) and with an emission filterof the 650EFLP type (Omega Optical).
 3. The method of claim 2, whereinthe optical filters exhibit the following qualities: they are of the6-cavity type; they have an ADI of 0°; they have a tolerance λ₀+20% ofFWHM; they have a tolerance on FWHM of ±20% of FWHM; they have an OD5out-of-passband rejection of UV at 1200 nm; they have a transmissioncurve T≧50% at λ₀.
 4. The method of claim 3, wherein the optical filtersexhibit, in addition, the following characteristics: they have acentered useful diameter greater than 21 mm; they have a thickness ≦7mm.
 5. The method of claim 2, wherein said multicolor FISH is akaryotype analysis.
 6. The method of claim 5, wherein said karyotypeanalysis is performed to detect chromosome rearrangements.
 7. A kitcomprising at least one fluorophore having a specific absorption andemission wavelength, said kit further comprising at least one pair ofoptical filters, said pair of optical filters comprising one absorptionfilter for detecting signals at said absorption wavelength and oneemission filter for detecting signals at said emission wavelength,wherein said at least one fluorophore, absorption filter, and emissionfilter are selected from the group consisting of: (a) the fluorophoreFITC having a maximum absorption wavelength of 494 nm and a maximumemission wavelength of 517 nm is coupled with the excitation filter ofthe 490DF30 type (Omega Optical) and with an emission filter of the530DF30 type (Omega Optical); (b) the fluorophore Cy3 having a maximumabsorption wavelength of 554 nm and a maximum emission wavelength of 568nm combined with an excitation filter of the 546DF10 type (OmegaOptical) and with an emission filter of the 570DF10 type (OmegaOptical); (c) the fluorophore TR having a maximum absorption wavelengthof 593 nm and a maximum emission wavelength of 613 nm combined with anexcitation filter of the 590DF10 type (Omega Optical) and with anemission filter of the 615DF10 type (Omega Optical); (d) the fluorophoreCy5 having a maximum absorption wavelength of 652 nm and a maximumemissions wavelength of 670 nm combined with an excitation filter of the650DF20 type (Omega Optical) and with an emission filter of the 670DF10type (Omega Optical); (e) the fluorophore Cy5.5 having a maximumabsorption wavelength of 675 nm and a maximum emission wavelength of 694nm combined with an excitation filter of the 680DF20 type (OmegaOptical) and with an emission filter of the 700EFLP type (OmegaOptical); and (f) the fluorophore Bodipy 630/650 having a maximumabsorption wavelength of 632 nm and a maximum emission wavelength of 658nm used with an excitation filter of the 630DF20 type (Omega Optical)and with an emission filter of the 650EFLP type (Omega Optical).
 8. Thekit according to claim 7, wherein said kit further comprises thefluorophore Cy7 having a maximum absorption wavelength of 743 nm and amaximum emissions wavelength of 767 nm combined with an excitationfilter of the 740DF25 type (Omega Optical) and with an emission filterof the 780EFLP type (Omega Optical).
 9. The kit according to claim 7,wherein said kit comprises: (a) the fluorophore Cy3 having a maximumabsorption wavelength of 554 nm and a maximum emission wavelength of 568nm combined with an excitation filter of the 546DF10 type (OmegaOptical) and with an emission filter of the 570DF10 type (OmegaOptical); (b) the fluorophore TR having a maximum absorption wavelengthof 593 nm and a maximum emission wavelength of 613 nm combined with anexcitation filter of the 590DF10 type (Omega Optical) and with anemission filter of the 615DF10 type (Omega Optical); and (c) at leastone fluorophore, absorption filter, and emission filter are selectedfrom the group consisting of: (i) the fluorophore FITC having a maximumabsorption wavelength of 494 nm and a maximum emission wavelength of 517nm is coupled with the excitation filter of the 490DF30 type (OmegaOptical) and with an emission filter of the 530DF30 type (OmegaOptical); (ii) the fluorophore Cy5 having a maximum absorptionwavelength of 652 nm and a maximum emissions wavelength of 670 nmcombined with an excitation filter of the 650DF20 type (Omega Optical)and with an emission filter of the 670DF10 type (Omega Optical); (iii)the fluorophore Cy7 having a maximum absorption wavelength of 743 nm anda maximum emissions wavelength of 767 nm combined with an excitationfilter of the 740DF25 type (Omega Optical) and with an emission filterof the 780EFLP type (Omega Optical); (iv) the fluorophore Cy5.5 having amaximum absorption wavelength of 675 nm and a maximum emissionwavelength of 694 nm combined with an excitation filter of the 680DF20type (Omega Optical) and with an emission filter of the 700EFLP type(Omega Optical); and (v) the fluorophore Bodipy 630/650 having a maximumabsorption wavelength of 632 nm and a maximum emission wavelength of 658nm used with an excitation filter of the 630DF20 type (Omega Optical)and with an emission filter of the 650EFLP type (Omega Optical).
 10. Thekit according to claim 7, wherein said kit comprises at least two ofsaid fluorophores and at least two pair of said optical filters.
 11. Thekit according to claim 7, wherein said kit comprises at least three ofsaid fluorophores and at least three pair of said optical filters. 12.The kit according to claim 7, wherein said kit comprises at least fourof said fluorophores and at least four pair of said optical filters. 13.The kit according to claim 7, wherein said kit comprises at least fiveof said fluorophores and at least five pair of said optical filters. 14.The kit according to claim 10, wherein said kit further comprises thefluorophore Cy7 having a maximum absorption wavelength of 743 nm and amaximum emissions wavelength of 767 nm combined with an excitationfilter of the 740DF25 type (Omega Optical) and with an emission filterof the 780EFLP type (Omega Optical).
 15. The kit according to claim 11,wherein said kit further comprises the fluorophore Cy7 having a maximumabsorption wavelength of 743 nm and a maximum emissions wavelength of767 nm combined with an excitation filter of the 740DF25 type (OmegaOptical) and with an emission filter of the 780EFLP type (OmegaOptical).
 16. The kit according to claim 12, wherein said kit furthercomprises the fluorophore Cy7 having a maximum absorption wavelength of743 nm and a maximum emissions wavelength of 767 nm combined with anexcitation filter of the 740DF25 type (Omega Optical) and with anemission filter of 780EFLP type (Omega Optical).
 17. The kit accordingto claim 13, wherein said kit further comprises the fluorophore Cy7having a maximum absorption wavelength of 743 nm and a maximum emissionswavelength of 767 nm combined with an excitation filter of the 740DF25type (Omega Optical) and with an emission filter of the 780EFLP type(Omega Optical).